1. Field of the Invention
The present invention is directed to a device, system, and method for improved detection of gas-phase conformations of, for example, protein-ligand complexes, functional macromolecular protein assemblies, and the like, and, more particularly, to a device, system, and method that use rapid deuterium labeling in a traveling-wave ion guide of a mass spectrometer performed alone or in tandem with ion mobility separation for improved detection.
2. Summary of the Related Art
Numerous studies have demonstrated that protein-ligand complexes and even large functional macromolecular protein assemblies can retain their non-covalent bonding when in the gas phase. This phenomenon enables the determination of stoichiometry and binding interactions by various gas-phase techniques such as limited collisional dissociation and ion mobility separation. In contrast, smaller globular proteins appear to adopt a multitude of gas-phase conformations depending on the condition of the electrospray process and the amount of time that elapses before detection. Although this conformational ensemble likely extends beyond that present in solution, gas-phase conformations of globular proteins offer a window into the non-native and solvent-free conformational landscape including intermediates along, for example, folding pathway and trapped misfolded species.
This information can be relevant for understanding important areas of biology such as protein folding, protein aggregation, and amyloid formation. Furthermore, several recent experimental studies suggest that solution-phase conformers of even small globular proteins can be largely preserved for 30-60 milliseconds following electrospray ionization (ESI). To take advantage of this phenomenon, sensitive analytical tools are needed for the rapid characterization of conformations of both small globular proteins and large macromolecular complexes in the gas phase.
Several techniques are available for interrogating the conformational properties of gaseous protein ions. These techniques include ion mobility spectrometry by which ions in an inert bath gas at high pressure are separated by drift-time and measurement of the kinetics of gas-phase chemistry such as proton transfer reactions, hydrogen/deuterium exchange (HDX), and the like. Although ion mobility spectrometry has proved an invaluable tool and has recently been introduced in commercially-available instruments, gas-phase HDX measurements provide an additional dimension for conformational interrogation that ion mobility spectrometry alone cannot provide.
Indeed, in a pioneering study by others, gas-phase HDX was used to provide some of the first experimental evidence of stable, coexisting, gas-phase, protein conformations. Other studies have shown that gas-phase HDX can sometimes expose the presence of additional gas-phase protein conformers not resolved by ion mobility spectrometry, and vice versa. Measuring the HDX of proteins in solution by mass spectrometry is an established method. Recent developments further enable the measurement of deuterium levels of individual amide hydrogen ions, similar to NMR spectroscopy. In contrast, mass spectrometric detection of gas-phase HDX has yet to see wide-spread use in biological research and the emerging field of native mass spectrometry. By combining conformation information obtained with solution HDX and those of gas-phase HDX experiments, it is possible to determine more definitively which conformations, present in the gas-phase shortly after ionization, are the same as those existing in solution.
In the field of mass spectrometry, chemical compounds can be ionized to generate charged molecules or molecule fragments from which their mass-to-charge ratios (m/z) can be measured, e.g., in a time-of-flight mass spectrometer (TOF-MS). Typically, mass spectrometers (MS) include an ion source, a mass analyzer, and a detector. The ion source converts molecules from a solution sample into ions, which are then sorted in the presence of an electromagnetic field according to mass by the mass analyzer. The detector measures the quantity of discrete ions present.
Isotopic labeling studies of gaseous proteins have typically been confined to mass spectrometers having custom-built ion traps/drift-tubes or Fourier transform-ion cyclotron resonance (FT-ICR) instruments. Ion traps use electric fields, e.g., a Paul trap, to capture ions and to determine their mass-to-charge ratio (M/z). A FT-ICR cell instrument uses a combination of electric and magnetic fields to trap ions in the confined volume of the ICR cell, e.g., a Penning trap, and determines the m/z value of ions based on the cyclotron frequency of ions in the fixed magnetic field. For gas-phase, isotopic labeling experiments in both ion traps or FT-ICR cells, a deuterated bath gas is introduced into the trap/cell so that the trapped molecules can be incubated in the presence of the bath gas for various periods of time.
Numerous gas-phase HDX studies have been performed using FT-ICR instruments in which ions are labeled while stored in an external RF-only ion guide or where ions are contained in the ICR cell. This enables defined ion-molecule reaction times from seconds to hours. Trapping ions in multipole-type ion reservoirs rather than in ICR cells facilitates the use of higher reagent gas-pressures and shorter gas-phase labeling times, e.g., less than 50 msec. The continuous accumulation of ions in an external ion reservoir during a gas-phase HDX reaction, however, can give rise to complex exchange kinetics as ions of the same origin are labeled for different amounts of time depending on their time of entry into the ion reservoir. Furthermore, filling the ion reservoir beyond its space charge limit can result in vibrational excitation and dissociation, which can further complicate interpretation of HDX kinetics. Notably, such issues have been addressed with custom-designed, gated-beam ESI sources having ion shutters and/or by using a MALDI source that does not produce continuous ion beams.
Accordingly, it would be desirable to provide a device, system, and method for performing gas-phase HDX labeling of the conformations of known or unknown ions in a traveling wave ion guide. Moreover, it would be desirable to accomplish this in tandem with ion mobility separation.